Karelle Leroy

Increased level of active GSK-3β in Alzheimer’s disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration

The somatodendritic accumulation of hyperphosphorylated tau proteins is an early event preceding the appearance of neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) and might be necessary for their formation. Glycogen synthase kinase‐3β (GSK‐3β) is a physiological kinase for tau that generates many tau phosphorylation sites identified in NFT and in other tau‐positive inclusions. We have studied the cellular distribution and the expression of the active form of GSK‐3β (GSK‐3 pTyr216) in AD patients, in argyrophilic grain disease and in diffuse Lewy body disease. By Western blotting analysis, a significant increase in the level of GSK‐3 (pTyr216) was observed in the frontal cortex of AD patients. A population of neurones showed a somatodendritic accumulation of GSK‐3 (pTyr216) but not of the inactive form of GSK‐3β (GSK‐3 pSer9). Most of these GSK‐3 (pTyr216)‐positive cells were positive for six different phosphotau epitopes known to be generated by GSK‐3β. By using a quadruple labelling method using GSK‐3 (pTyr216) and phosphotau immunolabelling combined with Gallyas and DAPI staining, we examined neurones containing a somatodendritic GSK‐3 (pTyr216) immunoreactivity at different stages of neurodegeneration. A majority of neurones at the pretangle stage without Gallyas‐positive inclusions were GSK‐3 (pTyr216) positive and this GSK‐3 (pTyr216) immunoreactivity remained in most cells containing Gallyas and phosphotau‐positive inclusions excepted in extracellular NFT. A GSK‐3 (pTyr216) immunoreactivity was present in argyrophilic grains but not in cortical Lewy bodies. These results directly suggest that the activity of GSK‐3β is increased in AD and that somatodendritic accumulation and activation of GSK‐3β is an early event preceding and accompanying the formation of NFT and of other tau‐positive inclusions.

Developmental expression and localization of glycogen synthase kinase-3β in rat brain

Glycogen synthase kinase (GSK)-3beta is a protein kinase in the wingless/wnt pathway and as such is involved in the regulation of growth and development of the neural tissue in Drosophila and in vertebrates. This enzyme is also abundantly expressed in the mammal adult brain, where it might play a role in the regulation of several substrates. The expression and the neuroanatomical distribution of GSK-3beta immunoreactivity in the rat brain from embryonic up to adult stages has been studied. GSK-3beta was expressed in the developing brain with the highest expression observed from 18 days of embryonic life up to 10 days of postnatal life. Its expression decreased thereafter and was lowest in the adult. GSK-3beta was strongly expressed in developing neurons but only weakly expressed in layers containing neuroblasts. In the adult and during development, GSK-3beta was detected in the pericarya and proximal part of dendrites. In the embryo, an intense GSK-3beta immunoreactivity was also observed in axonal tracts. This axonal immunoreactivity had markedly decreased by 10 days of postnatal life and was absent at 20 days of postnatal life and in the adult. No GSK-3beta immunoreactivity was detected in astrocytes. The GSK-3beta immunoreactivity was found in most brain regions, although significant local variations of GSK-3beta expression were observed. The developmental evolution of GSK-3beta compartmentalization in neurons parallels that of phosphorylated tau, a protein considered to be a physiological substrate for the kinase.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimers disease.

Deletion of Irs2 reduces amyloid deposition and rescues behavioural deficits in APP transgenic mice.

As impaired insulin signalling (IIS) is a risk factor for Alzheimer’s disease we crossed mice (Tg2576) over-expressing human amyloid precursor protein (APP), with insulin receptor substrate 2 null (Irs2−/−) mice which develop insulin resistance. The resulting Tg2576/Irs2−/− animals had increased tau phosphorylation but a paradoxical amelioration of Aβ pathology. An increase of the Aβ binding protein transthyretin suggests that increased clearance of Aβ underlies the reduction in plaques. Increased tau phosphorylation correlated with reduced tau-phosphatase PP2A, despite an inhibition of the tau-kinase glycogen synthase kinase-3. Our findings demonstrate that disruption of IIS in Tg2576 mice has divergent effects on pathological processes—a reduction in aggregated Aβ but an increase in tau phosphorylation. However, as these effects are accompanied by improvement in behavioural deficits, our findings suggest a novel protective effect of disrupting IRS2 signalling in AD which may be a useful therapeutic strategy for this condition.

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

Mint2/X11-like colocalizes with the Alzheimer’s disease amyloid precursor protein and is associated with neuritic plaques in Alzheimer’s disease

Aberrant metabolism of the amyloid precursor protein (APP) is believed to be at least part of the pathogenic process in Alzheimers disease. The carboxy‐terminus of APP has been shown to interact with the Mint/X11 family of phosphotyrosine binding (PTB) domain‐bearing proteins. It is via their PTB domains that the Mints/X11s bind to APP. Here we report the cloning of full‐length mouse Mint2 and demonstrate that in primary cortical neurons, Mint2 and APP share highly similar distributions. Mint2 also colocalizes with APP in transfected CHO cells. In Mint2/APP‐cotransfected cells, Mint2 reorganizes the subcellular distribution of APP and also increases the steady‐state levels of APP. Finally, we demonstrate that Mint2 is associated with the neuritic plaques found in Alzheimers disease but not with neurofibrillary tangles. These results are consistent with a role for Mint2 in APP metabolism and trafficking, and suggest a possible role for the Mints/X11s in the pathogenesis of Alzheimers disease.

Lithium Treatment Arrests the Development of Neurofibrillary Tangles in Mutant Tau Transgenic Mice with Advanced Neurofibrillary Pathology

Neurofibrillary tangles (NFTs) made of phosphorylated tau proteins are a key lesion of Alzheimers disease and other neurodegenerative diseases, and previous studies have indicated that lithium can decrease tau phosphorylation in tau transgenic models. In this study, we have reassessed the effectiveness of treatment per os with lithium on the prevention, the arrest, or the reversal of NFT development in a tau transgenic line (Tg30tau) developing severe neurofibrillary pathology in the brain and the spinal cord. Wild-type and Tgtau30 mice were treated per os with lithium carbonate or with natrium carbonate by chronic chow feeding for 8 months starting at the age of 3 months (to test for a preventive effect on NFT formation) or by oral gavage for 1 month starting at the age of 9 months (after development of NFTs). In mice treated by oral gavage, a decrease of tau phosphorylation and of Sarkosyl-insoluble aggregated tau was observed in the brain and in the spinal cord. The density of NFTs identified by Gallyas staining in the hippocampus and in the spinal cord was also significantly reduced and was similar to that observed at the beginning of the lithium treatment. In these animals, the level of brain beta-catenin was increased probably as a result of its stabilization by glycogen synthase kinase-3beta inhibition. Despite this inhibitory effect of lithium on NFT development, the motor and working memory deficits were not significantly rescued in these aged animals. Chronic chow feeding with lithium did not alter the development of NFT. Nevertheless, this study indicates that even a relatively short-term per os treatment leading to high blood concentration of lithium is effective in arresting the formation of NFTs in the hippocampus and the spinal cord of a tau transgenic model.

Expression of tau mRNA and soluble tau isoforms in affected and non-affected brain areas in Alzheimer's disease.

In Alzheimers disease (AD), selective expression of tau isoforms might underlie the susceptibility of different brain areas to develop neurofibrillary tangles and this pattern might change in the disease. In this study, we have analyzed in control subjects and in sporadic AD patients the pattern of expression of tau mRNA and tau proteins in areas unaffected (cerebellar cortex, white matter), moderately affected (occipital striate cortex, thalamus, caudate nucleus, and putamen) or strongly affected by neurofibrillary tangles (temporal and frontal associative cortex). After RT‐PCR amplification, five products corresponding to the tau mRNAs containing exons 2 and 3, exon 2, without exons 2 or 3, with exon 10 and without exon 10 were identified. In control subjects, these five PCR products were present in all areas except in white matter, where transcripts with exons 2 or exons 2 and 3 were not identified. In AD patients, the same pattern of transcripts was observed in different areas, regardless of the presence of neurofibrillary lesions. After dephosphorylation of soluble tau proteins, the six tau isoforms were identified in the same areas by immunoblotting, including in the white matter, suggesting that most tau isoforms with exons 2 and 3 are transported along axons. The relative expression of 0N3R isoforms was higher in the temporal cortex than in the cerebellar cortex, both in control and AD subjects. The qualitative pattern of expression was identical in subjects with or without an APOE4 allele. Our results suggest that splicing regulation of the tau gene and the relative expression of tau isoforms are not significantly changed in sporadic cases of the disease, although differential expression of tau isoforms in temporal cortex might underlie this brain area susceptibility to neurofibrillary tangles formation.

Lack of Tau Proteins Rescues Neuronal Cell Death and Decreases Amyloidogenic Processing of APP in APP/PS1 Mice.

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ.

Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury

Respiratory dysfunction is the leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). Injuries targeting mid-cervical spinal cord regions affect the phrenic motor neuron pool that innervates the diaphragm, the primary respiratory muscle of inspiration. Contusion-type injury in the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. In an attempt to target the phrenic motor neuron pool, two unilateral contusion injury paradigms were tested, a single injury at level C4 and a double injury both at levels C3 and C4, and animals were followed for up to 6 weeks post-injury. Both unilateral cervical injury paradigms are reproducible with no mortality or need for breathing assistance, and are accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, changes in spontaneous diaphragm EMG recordings, and reduction in phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant and chronically persistent respiratory compromise following mid-cervical SCI due to phrenic motor neuron degeneration. These injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically relevant cervical SCI models.